7-Substituted 8-aza-7-deazaadenosines for modification of the siRNA major groove

Department of Chemistry, University of California, Davis, One Shields Avenue, Davis, California 95616, USA.
Organic & Biomolecular Chemistry (Impact Factor: 3.56). 07/2012; 10(32):6491-7. DOI: 10.1039/c2ob25647a
Source: PubMed


Here we describe the synthesis of new 7-substituted 8-aza-7-deazaadenosine ribonucleoside phosphoramidites and their use in generating major groove-modified duplex RNAs. A 7-ethynyl analog leads to further structural diversification of the RNA via post-automated RNA synthesis azide-alkyne cycloaddition reactions. In addition, we report preliminary studies on the effects of eight different purine 7-position modifications on RNA duplex stability and pairing specificity. Finally, the effect on RNAi activity of this type of modification at eight different positions in an siRNA guide strand has been explored. Analogs were identified with large 7-position substituents that maintain adenosine pairing specificity and are well-tolerated at specific positions in an siRNA guide strand.

13 Reads
  • [Show abstract] [Hide abstract]
    ABSTRACT: 8-Alkoxyadenosines have the potential to exist in anti or syn conformations around the glycosidic bond when paired opposite to U or G in the complementary strands, thereby placing the sterically demanding 8-alkoxy groups in the major or minor groove, respectively, of duplex RNA. These modified bases were used as "base switches" in the guide strands of an siRNA to prevent off-pathway protein binding during delivery via placement of the alkoxy group in the minor groove, while maintaining significant RNAi efficacy by orienting the alkoxy group in the major groove. 8-Alkoxyadenosine phosphoramidites were synthesized and incorporated into the guide strand of caspase 2 siRNA at four different positions: two in the seed region, one at the cleavage junction, and another nearer to the 3'-end of the guide strand. Thermal stabilities of the corresponding siRNA duplexes showed that U is preferred over G as the base-pairing partner in the complementary strand. When compared to the unmodified positive control siRNAs, singly modified siRNAs knocked down the target mRNA efficiently and with little or no loss of efficacy. Doubly modified siRNAs were found to be less effective and lose their efficacy at low nanomolar concentrations. SiRNAs singly modified at positions 6 and 10 of the guide strand were found to be effective in blocking binding to the RNA-dependent protein kinase PKR, a cytoplasmic dsRNA-binding protein implicated in sequence-independent off-target effects.
    Journal of the American Chemical Society 10/2012; 134(42):17643-52. DOI:10.1021/ja307102g · 12.11 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Short interfering RNAs (siRNAs) are promising drug candidates for a wide range of targets including those previously considered "undruggable". However, properties associated with the native RNA structure limit drug development and chemical modifications are necessary. Here we describe the structure-guided discovery of functional modifications for the guide strand 5' end using computational screening with the high resolution structure of human Ago2, the key nuclease on the RNA interference pathway. Our results indicate the guide strand 5'-end nucleotide need not engage in Watson-Crick (W/C) H-bonding but must fit the general shape of the 5'-end binding site in MID/PIWI domains of hAgo2 for efficient knockdown. 1,2,3-Triazol-4-yl bases formed from the CuAAC reaction of azides and 1-ethynylribose, which is readily incorporated into RNA via the phosphoramidite, perform well at the guide strand 5'-end. In contrast, purine derivatives with modified Hoogsteen faces or N2 substituents are poor choices for 5'-end modifications. Finally, we identified a 1,2,3-triazol-4-yl base incapable of W/C H-bonding that performs well at guide strand position 12, where base pairing to target was expected to be important. This work expands the repertoire of functional nucleotide analogs for siRNAs.
    Journal of the American Chemical Society 10/2013; 135(45). DOI:10.1021/ja4079754 · 12.11 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Among the contingency of methodologies available for bioconjugation, the Click dipolar [3+2] cycloaddition pathway that results in the formation of triazole linkage were extensively investigated during the last few years in a wide variety of molecular architectures -that ranges between small molecules and polymers. Synthetic feasibility as well as the stability and application of triazole linkage were explored and successfully accomplished in live cells, in vitro, in vivo and tissues. The objective of this review is to present recent reports of this synthetic methodology, as applied to oligonucleotide chemistry. Advances in development of Click solid supports, newer alkyne and azide building blocks, covalent conjugations at either termini (3′, 5′), internal sites (base, sugar and backbone), solid and solution-phase labeling strategies, and applications to oligonucleotide detection are summarized in this review.
    ChemInform 01/2014; 45(2). DOI:10.1002/chin.201402232
Show more